Mol Genet Genomics (2001) 266: 72±78
DOI 10.1007/s004380100520
O R I GI N A L P A P E R
E. GeÂrard á E. Jolivet á D. Prieur á P. Forterre
DNA protection mechanisms are not involved in the radioresistance
of the hyper thermophilic archaea P yrococcus abyssi and P. furiosus
Received: 1 February 2001 / Accepted: 4 May 2001 / Published online: 20 June 2001
Ó Springer-Verlag 2001
Abstract Hyperthermophilic archaea of the genus
Pyrococcus are resistant to gamma radiation, suggesting
that ecient mechanisms for DNA repair exist in these
organisms. To determine whether protective mechanisms
might also be implicated in this radioresistance, we have
estimated the linear density of DNA double-stranded
breaks caused by gamma irradiation in the genomic
DNA of two Pyrococcus species, using Escherichia coli
and the radioresistant bacterium Deinococcus radiodu-
rans as controls. The linear density of double-stranded
breaks was essentially the same in all four microorgan-
isms when irradiation was carried under similar
anaerobic conditions, indicating that no speci®c DNA
protection mechanisms exist in Pyrococcus species.
Using one- and two-dimensional gel electrophoresis we
compared the protein patterns from Pyrococcus abyssi
and P. furiosus cells that had or had not been exposed to
gamma rays. We did not detect any signi®cant protein
induction following DNA damage in either species.
Keywords Hyperthermophile á Archaea á
Radioresistance á DNA double-stranded breaks
Introduction
The phylogenetic domain Archaea includes many or-
ganisms that are speci®cally adapted to extreme
Communicated by R. Devoret
E. GeÂrard (&) á P. Forterre
Institut de GeÂneÂtique et Microbiologie, BaÃt. 409,
CNRS, UMR 8621, Universite Paris-Sud,
91405 Orsay Cedex, France
E-mail: egerard@ibp.u-psud.fr
Tel.: +33-1-69153346
Fax: +33-1-69153423
E. Jolivet á D. Prieur
IUEM/UBO, Technopole Brest-Iroise,
Place Nicolas Copernic, CNRS, UMR 6539,
29280 PlouzaneÂ, France
environments. They are able to grow at low pH, in high
salt concentrations or at high temperatures (Woese et al.
1990). Pyrococcus abyssi and P. furiosus are two hyper-
thermophilic archaea that are found in geothermal
habitats. They grow optimally at 100°C and 95°C,
respectively (Fiala and Stetter 1986; Erauso 1993). These
extremely high temperatures accelerate the spontaneous
degradation of DNA. The main problem with respect to
DNA stability at high temperatures is thermal degrada-
tion via depurination and subsequent breakage of the
phosphodiester bonds (Marguet and Forterre 1994). This
suggests that speci®c mechanisms might exist in hyper-
thermophilic archaea that protect and repair DNA. The
thermal degradation of DNA in P. furiosus has been
compared to that of Escherichia coli by estimating the
number of DNA backbone breaks after incubation of the
cells at 105°C. P. furiosus DNA was found to be 20 times
more resistant to thermal degradation than E. coli DNA
(Peak et al. 1995). The authors of that study observed that
some unidenti®ed proteins remained tightly bound to the
DNA. They suggested that the resistance of DNA to
thermal degradation could be partly due to proteins that
protect the DNA by limiting its contact with water. Fur-
thermore, DiRuggiero and collaborators (1998) reported
that P. furiosus could fully repair double-stranded DNA
breaks induced in its chromosome by exposure to
2500 Gy of gamma radiation, suggesting that this
organism possesses very ecient DNA repair systems.
Several years ago, Kopylov and collaborators (1993)
reported that two hyperthermophilic archaea,
Desulfurococcus amyloliticus and Thermococcus stetteri,
were radioresistant. They compared the radioresistance
of these organisms with those of the model bacterium
Escherichia coli and the bacterium Deinococcus radio-
durans, which is the most radioresistant organism
known. At doses that provoke the death of 50% or 90%
of the cells, D. amyloliticus and T. stetteri were 12±25
times more radioresistant than E. coli, and only about
three times less resistant than D. radiodurans (Kopylov
et al. 1993). P. furiosus is also particularly radioresistant.
This archaeon can withstand 2000 Gy of gamma

radiation without loss of viability, whereas the viability
of E. coli begins to decrease at doses of 100 Gy (DiR-
uggiero et al. 1997). Thus, radioresistance seems wide-
spread in the archaeal domain, since both Euryarchaea
(Thermococcus, Pyrococcus) and Crenoarchaea (Desulf-
urococcus) show this elevated radioresistance. However,
such a high level of radioresistance has only been re-
ported for hyperthermophilic archaea. Radioresistance
is not a common feature of all thermophilic organisms,
as the thermophilic bacteria Thermotoga maritima and
Thermodesulfobacterium are not radioresistant (Kopylov
et al. 1993).
D. radiodurans is resistant to a wide range of geno-
toxic agents, such as gamma radiation, UV radiation
and mitomycin C. Ecient repair of DNA damage is in
large part responsible for the resistance to genotoxic
agents (Battista 1999). As DNA is commonly considered
to be the critical cellular target of radiation, the radio-
resistance of Pyrococcus could be related, as in
D. radiodurans, to a high capacity for ecient DNA
repair (DiRuggiero et al. 1997). However, in contrast to
D. radiodurans, hyperthermophilic archaea are not par-
ticularly resistant to UV irradiation (Kopylov et al.
1993). Gamma radiation induces a wider variety of
DNA lesions than UV radiation (Friedberg et al. 1995).
Thus, gamma radiation causes lesions in both the bases
and the sugar residues of DNA, and results in the for-
mation of DNA strand breaks, whereas UV radiation
(254 nm) provokes mainly lesions in the DNA bases
(Friedberg et al. 1995). Thus, it seems unlikely that
hyperthermophilic archaea are able selectively to repair
only the lesions due to gamma radiation. During gamma
irradiation direct ionisation of DNA and an indirect
e€ect due to water radiolysis are involved in the for-
mation of DNA lesions (Ward 1998). The hydroxyl
radical intermediates formed during water radiolysis are
thought to cause 65% of the cell death (Ward 1998).
This indirect e€ect, due to hydroxyl radicals, does not
arise in cells irradiated with UV (Friedberg et al. 1995).
Thus, hyperthermophilic archaea could be speci®cally
resistant to the indirect e€ect of gamma irradiation
because they possess mechanisms that protect DNA
against hydroxyl radicals. Indeed both radiosensitising
agents and radioprotectants are known. Soluble intra-
cellular compounds such as thiols (Ward 1998) and
DNA-bound proteins (Ljungman et al. 1991, Boubrik
and Rouviere-Yaniv 1995) are thought to protect the
DNA against the damaging e€ects of ionising radiation.
In contrast, oxygen acts as a radiosensitising agent that
®xes lesions which could have been repaired in its ab-
sence (Ward 1990; Spotheim-Maurizot 1991). As some
proteins may be strongly attached to the DNA of
P. furiosus (Peak et al. 1995), DNA protection in hy-
perthermophilic archaea could be due to proteins that
limit the accessibility of DNA to hydroxyl radicals. In
agreement with this hypothesis, Isabelle and collabora-
tors (1993) have shown that the protein MC1 of the
hyperthermophilic archaeon Methanococcus sp. CHTI55
can protect against the radiolysis of DNA by fast
73
neutrons in vitro. This protein, which is known to
protect DNA against thermal denaturation, binds in a
non-speci®c fashion, and induces bending, supercoiling
and compaction of DNA (Isabelle et al. 1993).
To obtain insight into the mechanisms of DNA
protection, we analyzed the radioresistance of P. abyssi
and P. furiosus, whose genomes have recently
been completely sequenced (Maeder et al. 1999;
www.Genoscope.fr). For this, we compared, under
similar conditions, the radioresistance of P. abyssi with
that of the bacteria E. coli and D. radiodurans. We then
measured the linear density of double-stranded breaks
(DSBs) resulting from gamma irradiation of the genomic
DNA of these three organisms and of P. furiosus using
pulsed-®eld agarose gel electrophoresis. The linear den-
sities were similar in all these organisms, indicating that
the radioresistance of Pyrococcus species is not due to
speci®c protection of DNA but rather to an ecient
DNA repair system. Under our anaerobic conditions
Escherichia coli was more radioresistant than previously
reported (Kopylov et al. 1993; Shahmohammadi et al.
1997). This work thus shows that the conditions of
irradiation are crucial when the radioresistances of
di€erent organisms are being compared. In our experi-
ments, we found no clear evidence for signi®cant
induction of any particular protein by gamma radiation,
using two-dimensional gel electrophoresis of radioac-
tively labelled proteins. In contrast, proteins synthesised
in response to gamma irradiation are detectable in E. coli
(West and Emmerson 1977) and D. radiodurans (Hansen
1980; Tanaka et al. 1996). Therefore, the method we
used appears not to be suciently sensitive to identify
proteins induced in response to gamma irradiation in
Pyrococcus species. Another possibility is that most
proteins involved in Pyrococcus radioresistance are ex-
pressed constitutively. Further investigations using other
methods must be performed to answer this question.
Materials and methods
Strains and media
Pyrococcus abyssi (Erauso 1993) and P. furiosus (Fiala and Stetter
1986) were grown in YPC medium (Yeast Peptone Cystine), which is
similar to YPS medium (Erauso 1993) except that the sulphur is
replaced by cystine. E. coli strain AB1157 and D. radiodurans RI were
kindly supplied by Adriana Bailone (Institut Curie, Orsay, France).
The E. coli strain was cultivated in Luria Broth (LB) medium or LB
agar. D. radiodurans was cultivated in enriched 2´TGY (1% tryp-
tone, 0.6% yeast extract, and 0.2% glucose) or on TGY agar.
Gamma irradiation of cells
The strains were irradiated at the end of the exponential growth
phase in YPC or in mineral medium (YPC without yeast extract,
peptone or cystine) under anaerobic conditions in Hungate tubes.
Air was removed applying a vacuum and replaced by saturating the
tubes with N2. Resazurin was added at 1 mg/l as a redox indicator
and Na2S was added at 0.1% to ensure complete anaerobiosis.
Cultures were irradiated at a rate of 60 Gy/min using a 137Ce c-ray
source (Institut Curie, Orsay, France). The number of viable
74

cells was estimated by plating serial dilutions, under anaerobic
conditions for Pyrococcus and aerobic conditions for E. coli and
D. radiodurans, in the appropriate growth medium. For E. coli and
D. radiodurans, the number of viable cells was also estimated after
plating of di€erent dilutions onto LB agar or TGY agar.
Pulsed-®eld agarose gel electrophoresis (PFGE)
After irradiation, the cells were washed in arti®cial sea water for
Pyrococcus or in 0.9% NaCl for E. coli and D. radiodurans, and
suspended in 0.125 M EDTA at a density of 5´108 cells/ml. The
suspensions were then mixed with low-melting-point agarose to
obtain a ®nal concentration of 0.8% agarose. The 300-ll agarose
blocks were then incubated overnight in ESP bu€er (0.5 M EDTA
pH 9±9.5, 1% lauroyl sarcosine, 1 mg/ml proteinase K) at 37°C.
One-®fth of each agarose block was washed once with TE bu€er
containing 1 mM phenylmethylsulfonyl ¯uoride and then four
times with TE bu€er. The intact chromosomal DNA contained in
the agarose plugs was digested with 20 U of NotI restriction
enzyme overnight at 37°C. Agarose plugs were analysed on 1%
agarose gels in 0.5´TBE using a CHEF-MAPPER electrophoresis
system (Bio-Rad) at 6.5 V/cm2 for 24 h at 16°C, with a linear pulse
ramp of 15±70 s and a switching angle of 120°C. The gels were
stained with water containing ethidium bromide (0.5 lg/ml) for
30 min and destained for 15 min in water. Gel images were digi-
tised with a Sony CDD video camera and analysed using the
GELSCAN software written by Yvan Zivanovic in our laboratory.
Estimation of the linear density of double-stranded DNA breaks
introduced by gamma irradiation
The linear density of DNA breaks was calculated using the fol-
lowing equation, where Pir is the proportion of each DNA band in
the irradiated extracts, P0 represents the proportion of the same
DNA band in the non-irradiated extract, L is the size of the DNA
band in bp and D is the dose of irradiation in Grays (Gy).
1 Pir
P0
LD
Two-dimensional gel electrophoresis of proteins
After irradiation, 2-ml aliquots of exponential-phase P. abyssi and
P. furiosus cells, at a density of 1.5´106 cells/ml, were grown in the
presence of [35S]methionine (80 lCi/ml) in YPC medium. The cells
were lysed in 40 ll of bu€er A [9.5 M urea (Promega), 2% CHAPS
(Sigma), 0.1% DTT (Boehringer Mannheim), 0.8% ampholytes
3±10 (ESA), and 8 mM PMSF (Sigma)] by three cycles of freezing
and thawing in liquid nitrogen. Linear immobilised pH 3±10 gra-
dient strips (13 cm, Pharmacia) were rehydrated in bu€er A con-
taining the proteins, mixed with 175 ll of Bu€er B [9 M urea
(Promega), 2% Chaps (Sigma), 0.23% DTT (Boehringer Mann-
heim), 0.8% ampholytes 3±10 (ESA), 0.04% bromophenol blue,
8 mM phenylmethylsulfonyl ¯uoride (Sigma)]. The proteins were
focused for 5.5±6.5 h at 3500 V. The proteins were then separated
on a 12% SDS-PAGE gel (Laemmli 1970). The autoradiographs of
the gels were obtained with a phosphoimager Image Quant
(Molecular Dynamics).
radiation dose under aerobic or anaerobic conditions, to
look at the e€ect of the presence of oxygen during
irradiation. Under anaerobic conditions, all the P. abyssi
cells survived a dose of up to 2000 Gy (Fig. 1). At higher
doses, viability decreased exponentially. As expected,
oxygen increases the radiosensitivity dramatically. The
doses of radiation allowing 37% survival (D37) in min-
eral medium are 2111 Gy under anaerobiosis and
305 Gy under aerobic conditions. The slope of the sur-
vival curve is 1.67 times steeper in the presence of oxy-
gen. In previous reports, oxygen was shown to increase
the radiosensitivity by a factor of 2.5±3 (Shenoy et al.
1975), whereas in our experiment, the D37 value is seven
times lower in the presence of oxygen. This observation
can be explained by the fact that oxygen kills P. abyssi,
which is a strict anaerobe, even in the absence of ionising
radiation (Erauso 1993). Furthermore, the Na2S added
to reduce the medium in the anaerobic cultures could
also have a radioprotective e€ect. The survival rate of
the cells is higher in organic medium than in mineral
medium at high doses of irradiation. This could be due
to a protective e€ect of the cystine ± which contains
sulphydryl residues ± in the organic medium.
We then compared the survival curve for P. abyssi
with those for D. radiodurans and E. coli after irradia-
tion under the same anaerobic conditions (Fig. 2). The
three strains were irradiated under a nitrogen atmo-
sphere in the presence of the reducing agent Na2S at a
concentration of 0.1%. The curves presented here are
based on the average survival values from two or three
independent experiments. P. abyssi was markedly less
radioresistant than D. radiodurans since the latter did
not exhibit loss of viability, at least up to a dose of
irradiation of 3000 Gy (the highest dose tested in our
experiments). [The clear correlation between the number
of DNA double-stranded breaks and the radiation doses
(see below) con®rms that D. radiodurans was actually
exposed to the radiation.] In contrast, P. abyssi is more

Results

Survival curves for P. abyssi, E. coli

and D. radiodurans after irradiation with gamma rays

We ®rst measured survival rates for P. abyssi after Fig. 1 Survival curves for P. abyssi irradiated with gamma rays
exposure to gamma radiation, as a function of the emitted by 137Ce under various conditions

Fig. 2 Survival curves for P. abyssi, D. radiodurans and E. coli
after exposure to gamma radiation from 137Ce
radioresistant than E. coli. The survival curve for E. coli
decreases exponentially at 100 Gy, the lowest dose
tested. The D37 values for P. abyssi and E. coli are
2436 Gy and 614 Gy, respectively. The slope of the ex-
ponential curve for P. abyssi, calculated from the values
shown in Fig. 1, is however 1.6 times steeper than that
constructed from the E. coli data. The level of radiore-
sistance of E. coli is higher than usually reported in the
literature for cultures irradiated in the absence of oxy-
gen. In the experiments reported by Kopylov et al.
(1993), 50% survival is obtained at a dose of 100 Gy,
whereas a radiation dose of 417 Gy is necessary to re-
duce survival by this amount under our conditions. The
di€erence could be due to the high level of reduction of
the culture medium obtained in our experiments by
addition of Na2S.
Number of DNA breaks induced
by gamma irradiation
To compare the linear density of DNA breaks between
the two Pyrococcus species, E. coli and D. radiodurans,
we analyzed their genomic DNAs after irradiation us-
ing PFGE. The genomic DNA isolated from irradiated
cells was digested with NotI before loading on the gel in
order to quantify the disappearance of bands with
discrete sizes. For each organism, induced DSBs in the
DNA were detected at doses of radiation as low as 500
or 1000 Gy (Fig. 3). As expected, the bands corre-
sponding to the longer DNA fragments disappeared
®rst, since there is a higher probability of ®nding a DSB
in such fragments. The linear densities of DSBs are
shown in Table 1. The results are averages of the values
obtained for each DNA fragment at each dose and,
except in the case of P. furiosus, two independent ex-
periments per species. In agreement with the impression
gained by visual inspection of the gel pro®les, values
obtained for all four species were very similar, varying
from 0.72´10±9 DSB/Gy/bp for P. furiosus to 1.09´10±9
DSB/Gy/bp for D. radiodurans. These results indicate
the absence of speci®c mechanisms that prevent the
formation of DSBs in the two Pyrococcus species.
75
Gamma-ray irradiation does not cause major changes
in protein patterns in Pyrococcus
We tried to ®nd induced proteins that might be involved
in radioresistance in Pyrococcus species by analysing
one- and two-dimensional gel pro®les of total protein
extracts from P. abyssi and P. furiosus after irradiation
of the cultures with 180±3000 Gy of gamma radiation.
In the ®rst set of experiments, the proteins were stained
with silver to look at the steady-state pattern of whole
protein extracts (not shown). We failed to detect any
signi®cant radiation-dependent changes in these pat-
terns. To examine the patterns of newly synthesised
proteins after irradiation, proteins were labelled with
[35S]methionine during growth for 10±105 min, and ex-
tracted at di€erent times after irradiation (from 30 min
to 6 h). The protein patterns remained essentially similar
with and without irradiation with doses of 180±720 Gy
(shown in Fig. 4 for 720 Gy). At higher doses, the
amount of newly synthesized proteins in irradiated cells
decreased in some experiments but the protein patterns
were conserved. Some proteins (indicated by arrows)
seem to be induced or to migrate di€erently after irra-
diation in the experiment shown in Fig. 4. However,
these di€erences in protein patterns were not reproduc-
ible. This result shows that there are no striking
modi®cations in protein patterns (relative induction or
repression) in P. furiosus or P. abyssi in response to
gamma-ray irradiation.
Discussion
It was previously reported that several hyperthermo-
philic archaea are more radioresistant than E. coli
but less resistant than D. radiodurans (Kopylov et al.
1993; DiRuggiero et al. 1997). We con®rmed this
observation here by comparing the e€ect of gamma
rays on the two latter organisms with that on two
Pyrococcus species, P. abyssi and P. furiosus, under the
same conditions.
An attractive hypothesis was that the radioresistance
of Pyrococcus was due to some adaptation to high
temperatures. The mechanisms that protect the DNA of
P. furiosus against thermal degradation (Peak et al.
1995) could also protect DNA against the hydroxyl
radicals produced during irradiation. We have shown
here, however, that the number of DSB caused by
gamma radiation in genomic DNA is similar in the two
Pyrococcus species, E. coli and D. radiodurans. Thus, the
strong radioresistance of Pyrococcus is not related to
speci®c protection of DNA. It appears therefore that the
mechanism that protects the DNA against thermal de-
gradation at 100°C does not prevent the formation of
DNA breaks during irradiation.
The stronger radioresistance of Pyrococcus compared
to E. coli should be partly related to the size of the
chromosomal DNA (1.8 Mb for Pyrococcus abyssi,
4.6 Mb for Escherichia coli) since fewer DNA lesions are
76

Fig. 3 Pulsed-®eld gel

electrophoresis of genomic
DNA from P. abyssi,
P. furiosus, D. radiodurans
and E. coli. The strains were
irradiated with 0±3000 Gy
gamma radiation. Total
DNA was digested by NotI
and analyzed by pulsed-®eld
gel electrophoresis. On the
gel at the top left, the lengths
of the chromosomes of
Saccharomyces cerevisiae are
indicated in comparison
with a NotI digest of
genomic DNA of P. abyssi

Table 1 Linear density of DNA double-stranded breaks produced calculated that about 2 and 4 DSBs were created per
by gamma irradiation in Pyrococcus furiosus, P. abyssi, Deinococ- chromosome for E. coli and P. abyssi, respectively, at the
cus radiodurans and Escherichia coli
D37 dose. However, this comparison probably underes-
Species Linear density of DSB/Gy/bpa timates the di€erence in radioresistance between E. coli
and Pyrococcus species. In contrast to E. coli, the two
Pyrococcus furiosus 0.72 (‹0.24) ´10±9
Pyrococcus abyssi 0.84 (‹0.23) ´ 10±9
Pyrococcus species possess systems that fully counteract
Deinococcus radiodurans 1.09 (‹0.40) ´10±9 the deleterious e€ects of radiation at dosages of up to
Escherichia coli 0.82 (‹0.35) ´ 10±9 2000 Gy. D. radiodurans shows 100% survival when
a
exposed to a dose of radiation equivalent to 3000 Gy,
The linear density of double-stranded DNA breaks was calculated which induces the formation of nearly 10 DSBs in its
as described in Materials and methods
chromosome. The capacity to repair DSB is thus higher
in D. radiodurans than in E. coli or Pyrococcus.
accumulated in Pyrococcus for a given dose of irradia- As in E. coli and D. radiodurans, DSBs may be
tion. Based on the numbers of DSBs observed following repaired by homologous recombination in Pyrococcus
irradiation and the size of the chromosomal DNA, we species. P. abyssi and P. furiosus contain several copies
 77

Fig. 4 Comparison of
two-dimensional gel pro®les
of newly synthesized
proteins from P. furiosus and
P. abyssi before and after
gamma ray irradiation.
After irradiation with
720 Gy of gamma rays or no
irradiation (C) the cells were
grown in the presence of
[35S]methionine. Total
protein extracts were
analyzed on two-dimension-
al gels. The arrows indicate
proteins that seem to be
induced in this experiment

of their chromosomes during the log phase and the
stationary phase of growth (Bernander, personal com-
munication). Furthermore, the P. abyssi genome encodes
several proteins similar to both the RecA and Rad51
proteins, which are implicated in homologous recombi-
nation in bacteria and eukarya, respectively (our un-
published observations). We did not ®nd any signi®cant
protein induction after gamma irradiation. One possi-
bility is that the proteins involved in the response to
gamma irradiation are inducible and that our method is
not sensitive enough to detect them. Indeed, the two-
dimensional gels of proteins allowed us to observe be-
tween 200 and 300 individual proteins. This represents
only a tenth of all the proteins of P. abyssi and P. furiosus.
A more sensitive method, such as microarray technology,
should thus be used to look further for genes and pro-
teins that are induced in response to gamma rays. An-
other possibility is that most proteins involved in the
response to gamma irradiation are constitutively
expressed in P. abyssi and P. furiosus. In agreement with
this hypothesis, it has already been observed that some
proteins involved in DNA repair are constitutively ex-
pressed in Pyrococcus species. The two RecA/Rad51
homologues present in P. furiosus, RadA and RadB,
are not induced by gamma rays or UV irradiation
(Komori et al. 2000). Since P. abyssi and P. furiosus are
hyperthermophilic organisms, they probably need an
ecient DNA repair system which is continuously
expressed in order to maintain the integrity of their
genetic information. However, this situation may be
more general in Archaea, since pretreatment of the two
archaean species Halobacterium halobium and Sulfolo-
bus solfataricus with low doses of hydrogen peroxide or
N-methyl-N-nitrosoguanidine does not increase their
survival when exposed to higher doses. This suggests
that no adaptive response to DNA damage exists in
these archaea (Praul and Taylor 1997). The develop-
ment of ecient genetic tools is now required to iden-
tify the mechanisms involved in DNA radioresistance in
hyperthermophilic archaea.
78
Acknowledgements This work was support by a grant from EDF
(RB-2000-27). Emmanuelle GeÂrard is grateful to the Association
pour la Recherche contre le Cancer (ARC) for grant support.
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